Project Summary/Abstract Chronic pain is the most common complaint of patients. Most chronic pain patients are resistant to therapy, in large part because the underlying pathophysiology of their chronic pain condition is unknown. The ultimate goal of this research program is to fill this critical gap. Pain is strongly modulated by the rostroventral medulla (RVM) that directly regulates the activity of nociceptive dorsal horn neurons. Prominent in RVM are serotonin- containing neurons. However, the role of these neurons in chronic pain remains controversial, with evidence for both pathological increases and decreases in 5HT output. Our exciting preliminary findings?using a model of chronic pain after chronic constriction injury of the infraorbital nerve (CCI-Pain)?may resolve this important controversy. We show that, in CCI-Pain, RVM-5HT neuronal activity is amplified, resulting in abnormally high release of 5HT in the caudal dorsal horn ? trigeminal nucleus (SpVc). This causes SpVc neurons to produce a barrage of after-discharges (ADs) that far outlast nociceptive stimuli, and that are considered a manifestation of chronic pain. The increased 5HT release also potentiates the strength of nociceptive inputs to SpVc neurons. Coupled with our previous demonstration that reducing 5HT levels in RVM suppresses ADs and blocks pain sensitization, we hypothesize that increased serotonergic drive from RVM causes hyperexcitability of dorsal horn neurons, which results in chronic pain. Aim I tests the hypothesis that amplified activity of 5HT-RVM neurons results in increased release of 5HT in SpVc and the development of chronic pain. We test the prediction that the electrophysiological activity of optogenetically- identified 5HT RVM ?> SpVc projection neurons is amplified in CCI-Pain. We will also use in vivo fast scanning voltammetry, and quantitative mass spectrometry, to test the prediction that CCI-Pain is associated with increased 5HT release in SpVc. Aim II tests the hypothesis that increased 5HT release is causally related to the development of chronic pain. We will test the prediction that in vivo optogenetic release of 5HT from RVM terminals in SpVc results in signs of sensory and affective pain, and that these signs are exacerbated by repeated 5HT release. We will also test the converse prediction, that optogenetic inhibition of these 5HT terminals results in relief from CCI-Pain. Aim III tests the hypothesis that amplified 5HT activity produces chronic pain by inducing abnormal ADs in dorsal horn neurons. We will test the prediction that in vivo optogenetic release of 5HT induces ADs in SpVc neurons of uninjured animals, and that optogenetic inhibition of 5HT release will suppress ADs in CCI-Pain animals. Aim IV tests the hypothesis that amplified 5HT activity produces chronic pain by potentiating primary afferent inputs to dorsal horn neurons. We will test the prediction that optogenetic release of 5HT in vitro evokes potentiation of trigeminal inputs to SpVc neurons. The predicted findings have novel translational relevance for the development of new pharmaceuticals to treat chronic pain.